Production Of Chlorella Vulgaris With High Lutein Content And Beverage Development

Lutein is a common natural carotenoid in plants.It has strong antioxidant capacity and is widely used in the fields of medicine,health care and food processing.However,as the demand for lutein in the market continues to increase,the traditional low-efficiency and high-consumption method of extracting lutein from marigolds is difficult to meet these needs.Therefore,it is important to find a plant that can replace marigold for lutein production and develop a culture protocol that is less costly and more efficient in terms of time and effort for the production of lutein.This topic takes Chlorella vulgaris as the research object,explores the conditions for high lutein production of Chlorella vulgaris,finds a method to reduce the cost of Chlorella vulgaris cultivation,and develops a drink of Chlorella vulgaris fermented emulsion.The main research contents are as follows:（1）Optimization of conditions for high lutein production in Chlorella vulgaris.In this paper,the effects of different culture modes,nitrogen sources,carbon to nitrogen ratio and nitrogen to phosphorus ratio on the growth and total chlorophyll,carotenoid,lutein and protein accumulation of Chlorella vulgaris were investigated.The results showed that,compared with heterotrophic culture,mixed culture of Chlorella vulgaris had a higher growth rate,and could synthesize more nutrients under the promotion of light;compared with ammonium acetate and urea,sodium nitrate was more conducive to the Material synthesis of Chlorella vulgaris;adjusting the carbon-nitrogen ratio of the medium to 12:1 and the nitrogen-phosphorus ratio to 10:1 can significantly improve the carbon-nitrogen metabolism efficiency of Chlorella vulgaris,and greatly improve the ability of Chlorella vulgaris to reduce sugar,total organic carbon and total Nitrogen utilization improves biomass productivity and the production of various nutrients including lutein.The final optimization results are culture mode:mixed culture;nitrogen source:sodium nitrate;carbon-nitrogen ratio:12:1;nitrogen-phosphorus ratio:10:1.Under these conditions,the final biomass,biomass productivity,growth yield,total chlorophyll yield,carotenoid yield,lutein yield,lutein content and protein yield of Chlorella vulgaris were 6.16 g/L and 1.01 g/L,respectively/d,1.60 g _biomass/g _TOC,137.40 mg/L,30.99 mg/L,13.56 mg/L,2.20 mg/g and 2.28 g/L.（2）Enzymatic hydrolysis of sweet sorghum leachate as a substitute for glucose in the culture of Chlorella vulgaris.The enzymatic hydrolysis of the sweet sorghum juice leachate was carried out and the optimum hydrolysis conditions were obtained as follows:45 min of hydrolysis with 0.1%sucrose enzyme;the concentration of reducing sugars increased from 58.37 g/L to 91.27 g/L;the concentration of fructose increased from 17.72 g/L to 35.61 g/L.The sweet sorghum juice leachate enzymatic hydrolysis was used as an alternative carbon source to glucose for the cultivation of Chlorella vulgaris.Chlorella vulgaris.in sweet sorghum medium lacking nitrogen and minerals were found to be unable to grow rapidly to accumulate biomass,while Chlorella vulgaris.cultured in sweet sorghum medium supplemented with nitrogen and minerals showed a stronger advantage over Chlorella vulgaris.cultured under optimal conditions in BG-11 medium,with an increase in total chlorophyll production,carotenoid production,lutein production and protein production by 18.02%,The total chlorophyll production,carotenoid production,lutein production and protein production increased by 18.02%,13.34%,35.95%and 20.36%,respectively.The final biomass,biomass productivity,growth yield,total chlorophyll production,carotenoid production,lutein production,lutein content and protein production of Chlorella vulgaris under these conditions were 5.48 g/L,0.90 g/L/d,1.99 g _biomass/g _TOC,164.27mg/L,38.91 mg/L,20.42 mg/L,3.73 mg/L and 2.73 mg/L,respectively,Transcriptomic analysis showed that Chlorella vulgaris cultured with sweet sorghum leachate enzymatic digest showed some upregulation of genes in the photosynthesis-antennal protein pathway,tricarboxylic acid cycle pathway,and cysteine and methionine metabolism pathway compared to the blue-green algal medium.The upregulation of genes in the photosynthesis-antennal protein pathway indicated that Chlorella vulgaris synthesized more antennal proteins to collect and transmit light energy,which increased the efficiency of light energy capture and promoted photosynthesis in Chlorella vulgaris.The metabolites of the pathway,such as oxaloacetate,citrate,pyruvate andα-ketoglutarate,can be converted into raw materials for photosynthetic pigments and protein synthesis through a series of enzymatic reactions.The upregulation of genes in the cysteine and methionine metabolic pathways suggests that the synthesis of methionine and cysteine is enhanced,as is the closely related sulfur metabolism,which also promotes pigment synthesis.An assessment of the cost of the medium showed that the total cost per kg of biomass for Chlorella vulgaris culture using sweet sorghum leachate enzymatic digest was 3.189 CNY,which was only 34.48%of the total cost for the BG-11+glu group（9.249 CNY）.The results indicate that the method of culturing Chlorella vulgaris with sweet sorghum as an exogenous carbon source instead of glucose is economically significant and the strategy can effectively reduce the cost of the culture medium for Chlorella vulgaris.（3）Production of Chlorella vulgaris emulsion.The process conditions for making Chlorella vulgaris fermentation emulsion were explored,and the best parameters for emulsion preparation were obtained:using brewer’s yeast to access the Chlorella vulgaris culture for 12 h can consume all the fructose in the medium and obtain a good deodorization effect,bringing freshness to the fermentation solution;using the high-pressure homogenization method to break the wall of Chlorella vulgaris for 8 min at40 MPa can obtain more than 98%cell fragmentation rate;adding 0.10%mono-diglyceride fatty acid ester and 0.15%sucrose fatty acid ester can obtain the best emulsification effect;When homogenizing the emulsion,0.10%mono-and di-glycerol fatty acid esters and 0.15%sucrose fatty acid esters were added to obtain the best emulsification effect,with zero emulsion index and over 98%stability coefficient;when blending,0.015%rosmarinic acid glycosides and 0.05%citric acid were used,and the addition amount of fermentation broth in the aqueous phase of the emulsion was adjusted to 10%to obtain the best sensory evaluation effect.Finally,we obtained Chlorella vulgaris fermentation emulsion with creamy white color,uniform texture and color,light fresh taste,delicate and soft taste,and harmonious sweet and sour taste.